Project Summary Sequences of neuronal activity are thought to underlie planning, preparation, and production of voluntary skilled behaviors. Dissecting how these premotor and motor sequences are functionally and synaptically integrated to support fluent, context-appropriate performance of natural behaviors is a major research challenge and a central goal of the BRAIN Initiative. The songbird premotor cortical structure HVC (letters used as proper name) has emerged as a prominent model system for studying how sparse neural sequences underlie the production of a precise, ethologically relevant behavior - birdsong. A single class of projection neurons in HVC is necessary for acute performance of birdsong. Yet, technical limitations associated with cell- type selective monitoring and manipulation of these neurons has hindered the ability to study how their neural sequences are functionally and synaptically integrated to support the planning, preparation and execution of behavior. We have developed cell-type specific methods for population calcium imaging and optogenetic manipulations in HVC and demonstrate their value in dissecting the functional and synaptic organization of this circuit in singing birds. Our preliminary results support a new model for HVC functional organization that will be tested in the four aims of this proposal. We will use calcium imaging, electrophysiological recordings and optogenetic manipulations in freely singing birds to test how diverse neural sequences in HVC underlie the planning, preparation and production of song. In addition, we will use single-cell optogenetics, calcium imaging, and circuit mapping methods to test the functional, synaptic and areal organization of this circuit. Drawing on a variety of cutting-edge approaches and the combined expertise of three scientific groups, this research aims to provide a new functional model for how diverse neural sequences are synaptically integrated to support fluent production of a voluntary skilled behavior.